Disease Resistance and Immune Response Genes in Cattle: Strategies for Their Detection and Evidence of Their Existence

Dim)ase Resistance and Immune Response Genes in Cattle: Strategiøs for Their Detection and Evidønce of Their Existence

HARRIS A. LEWlN 1 Laboratory of Immunogenetics

Department of Animal Sciences University of Illinois

Urbana 61801

ABSTRACT

The possibility of breeding or genetically engineering cattle for resistance to disease has tremendous potential for increasing the efficiency of milk and meat production. In cattle and other species, major genes that control humoral and cellular immune responses to a variety of antigens have been mapped to a chromosomal region known as the major histocompatibility complex. However, resistance or susceptibility to viral, bacterial, and parasitic diseases in noninbred species is often a complex phenotype, with age, stress, and physiologic status all being important factors in the outcome of infection. This paper reviews the function of major histocompatibility complex gene products and the relationship between polymorphism of these genes and infectious diseases. A discussion of strategies for detecung immune response genes and disease associations is presented, with particular reference to the problems and advantages of working with cattle. The present knowledge of the bovine major histocompatibility complex and its relationship to immune responsiveness and disease resistance are also reviewed, with special consideration given to enzootic bovine leukosis because of the significant relaåonship between alleles of the bovine lymphocyte antigen system and resistance

Received August 31, 1988. Accepted November 10, 1988. ~This work was supponed in pm by grants from the

United States Depanment of Agriculture (87-CRCR-1- 2292 and 87-CRCR-1-2546) and Project Number 20-0317 of the Agricuitural Experiment Station. College of Agricul- ture, University of Illinois at Urbana-Champaign.

or susceptibility to subelinical progression of bovine leukemia virus infecfion. Finally, potential applications of this research to geneUc improvement and animal health are considered.

INTRODUCTION

Are there genes that influence immune responsiveness and disease resistance in cattle? If such genes can be identified, can they be uff- lized for genetic improvement? Only recently, the technologies and funds have become available to answer these questions. Selective breeding, or molecular genetic manipulation for the purpose of increasing disease resistance, may play an important future role in reducing reli- ance on the use of anUbiotics and other poten- aally harmful feed additives.

The impetus to study the genetic control of immune responsiveness in cattle and other farm animals was provided over 25 yr ago by the discovery of "immune response" (lr) genes in guinea pigs and mice (11, 58). Soon alter these discoveries, it was shown that the quanUtative concentrations of antibodies produced by immunization with several chemically defined antigens were under the control of genes linked to the major histocompatibility complex [MHC; (24, 57)]. These findings, the relationship between MHC genes and allograft survival, and the associations between MHC and disease susceptibility [extensively described in (41)], provided the most important reasons for the explo- sion of research on the MHC that occurred in the 1970s and 1980s. In 1980, Jean Dausset, Baruj Benacerraf, and George Snell shared the Nobel prize in Medicine and Physiology for their pioneering work on the biology of the MHC. An important event in the history of immunology occurred in 1987 with the publica- tjon of the three-dimensional structure of an MHC-encoded molecule (14). The intricate re-

1989 J Dairy Sci 72:1334--1348 1334

SYMPOSIUM: GENETICS OF DISEASE RESISTANCE 1335

lationship of MHC polymorphism to MHC function was revealed in the elegant geometry of the class I molecule. In this symposium paper, the role of the major histocompatibility complex (MHC) in immune response and disease resistance is reviewed, and the potential applications of this research to dairy cattle breeding are discussed.

THE MAJOR HISTOCOMPATIBILITY COMPLEX

Genøtio Control of Immune Røsponeivenøss

The biochemistry, genetics, and function of MHC molecules have be.en thoroughly re- searched (41). The mttrine MHC, or H-2 complex, svans approximately 2000 kilobases (kb) of DNA on mouse chromosome 17 (34). The MHC of man, HLA, is located on the short arm of chromosome 6. Genes of the MHC are grouped into three major classes. The class I genes encode proteins that are 337 to 351 amino acids in length and have a relative molecular mass (Mr) of approximately 44,000 alter glycosylation in the Golgi apparatus. Class I heavy chains are noncovalently bound to 132- microglobulin, which is encoded on chromosome 2 in the mouse and chromosome 15 of humans. The primary immunological function of class I molecules is to serve as "restriction elements" for CD8 ÷ cytotoxic T lymphocytes (CTL); i.e., the receptor for anagen on CTL recognizes that antigen only in associaaon with a MHC class I gene product (93). Class I- restricted CTL are important components of antiviral and antitumor effector responses.

The class II or lr genes encode the immune- associated (la) molecules. The class II genes were first named "Ir genes" because they determined the concentration of serum antibodies in response to immunization with simple synthetic antigens. The Ir genes were later mapped in the middle of the H-2 complex, between the H-2K and H-2D loci (41). Class II molecules are glycoprotein heterodimers, composed of an tx- chain (Mr 34,000) and 13-chain (Mr 28,000), which are encoded by tightly linked clusters of genes (41). Both chains consist of 229 to 238 amino acids, depending on the locus, with the difference in Mr due to glycosylation of the «- chain. Class II molecules function primarily as restriction elements for CD4 ÷ helper/inducer T-

cells; i.e., receptors on CD4 ÷ cells recognize antigen in association with class II molecules (69). In the inductive phase of an immune response, antigen is thought first to be internal- ized by an antigen presenting cell (dendritic cells, macrophages and B cells), and proteolytically degraded or "processed" (2) either by proteases at the cell surface or in the hydrolytic environment of endosomal vesicles (91). Processed peptides are bound to low affinity binding sites on class II molecules inside the cell and transported to the cell sufface where they are "presented" to antigen specific CD4 + helper T-cells. Recognition of the antigen-MHC class II complex by helper T-cells results in activation, proliferafion and elaboration of lym- phokines (38). These activated T-cells function either to enhance or suppress an immune response via the soluble mediators or by cell-ceU contact (41).

The class III region of the MHC contains the structural genes for serum complement components C2, C4, and factor B, and steroid 21- hydroxylase (34). It seems certain that the relatively large genetic distance between the class III and class I regions contains new genes soon to be discovered because of the availability for sequencing of ordered cosmid clones that span the MHC. The class III genes and other nonclass I and nonclass II genes located within the MHC, such as the tumor necrosis factor tx and ~1 genes, will not be considered further in this article. Although these genes play an important role in immunologic and metabolic diseases and may have important effects on quantitative phenotypes (41), they are not related in structure or function to the MHC class I and class II molecules.

Structure Function Relationøhips

The three-dimensional structure of a human class I molecule (HLA-A2) has recently been determined by X-ray crystallographic analysis (14). The dass I molecule has a binding crevice for an antigen fragment(s) that is formed by two (x helices joined by a "floor" of antiparaUel ~l-pleated protein strands (15). Antigen can be visualized to sit in the binding crevice like a "hot dog in a bun". Polymorphic amino acid residues, critical for antigen binding and T cell recognition, are located mainly on the in-

Journal of Dalry Science Vol. 72, No. 5, 1989

1336 LEWIN

ward face of the floor and aiong regions of tx- helices that face inward and up from the antigen binding sites. A similar structure has recently been predicted for class U molecules (18).

In light of the recent X-ray crystaUography studies and other experimentai data, lr gene phenomena observed in inbred mouse strains with synthetic antigens and peptide fragments can now be explained in biochemicai terms. Nonrespønsiveness to an antigen results if the spectrum of MHC class II gene products of a given strain are unable to bind an immunologicaily relevant peptide of that antigen (22). Al- ternatively, nonrespønsiveness can be a consequence of "blind spots" in the T ceU repertoire for a parficular antigen plus self MHC combi- nation (28). Such "blind spots" are thought to arise through the process of T-cell "education" to self MHC in the thymus. Current dogma argues that T-cells with a high affinity for self MHC, or for self MHC associated with endoge- nous self peptides, are deleted or inactivated (tolerance to seil'), whereas T-cells with lower affinity for self MHC are ailowed to differenti- ate and exit the thymus (39). Molecular mimicry between self-MHC complexed with endog- enous self peptide and a foreign antigen results in the so-caUed "blind spøt". In addiåon to MHC pølymorphism, other factors, such as quantitative MHC expression, might aiso influence the induction and magnitude of an immune respønse (35).

Unlike the synthetic antigens used to study Ir genes, bacteria, viruses, and parasites are antigenicaily complex. Interestingly, MHC- linked Ir genes exist for immunologicaily relevant epitopes on a number of pathogenic organisms (41). Hence, disease susceptibility could result if immunologicai unresponsiveness were to exist in an individuai for an important epi- tope(s) on a pathogenic agent. Although susceptibility to severai infectious diseases is influenced to a major extent by the MHC, most experimental evidence indicates susceptibility to infectious diseases is a complex phenotype that involves many genes (71). The topic of MHC-associated susceptibility to noninfectious diseases (e.g., autoimmune diseases, nonvirai cancers, metabolic diseases) is beyond the

scope of this article and the interested reader is referred to other sources (83).

Origin and Slgnlflcance of Major Histocompatlblllty Compløx Polymorphism

There are more than 100 alleles at the murine and human class I and class II loci, and MHC genes are highly polymorphic in nearly every other species so far studied (41). To gain an understanding of the role of the MHC in resistance and susceptibility to infectious diseases, a good deai of scientific and intellectuai effort has be, en applied to delineating and ratio- naiizing the extent of MHC polymorphism.

Biochemicai and molecular data suppon the hypothesis that the origin of MHC polymorphism predates speciation events (9, 56). The polymorphism of class I and class II genes is generated by point mutation, gene duplication, and gene conversion events. The latter genetic mechanism produces sequence variation through the exchange of blocks of genetic information from donor to recipient genes. The apparent exchange of a sequence of 13 nucleo- tides from a class I Qa region gene of the mouse into the homologous region of an H-2K gene is generaily accepted as evidencefor an operational gene conversion-like mechanism (69). Data supporting gene conversion-like events have aiso been presented for class II sequences (59).

The most likely explanafion for the extreme polymorphism at some MHC loci is naturai selection (16). Selectionist models argue that MHC polymorphism favors the survival of a species by reducing the risk of extinction by epidemic microbial infections. Although it is difficult to directly prove this hypothesis, there is a good deal of supporting data derived from gene frequency studies of human pøpulations (31) and nucleotide sequence anaiyses. From a detailed study of human class II sequences, Gustaffson et ai. (29) concluded that there is negative selection against mutations in the regions of class II genes that are important for functionai integrity of the molecule, whereas mutations in domains responsible for antigen binding appear to be "neutral". This permitted diversification in the antigen binding domain would lead to an expanded Ir gene repertoire.

Journal of Dalry Scieace Vol. 72, No. 5, 1989


The multimde of MHC alleles may thus provide a species-wide solution for the immunological dilemma of having to respond to a wide array of existing and potenUal pathogens using a relatively small number of genes.

Why should such a large number of alleles exist if only two aUeles per locus can be expressed by an individual? Why has natural selecfion not resulted in ftLrther expansion of the number of MHC loci? The number of MHC genes does vary in some haplotypes within a species (copy nurnber polymorphism). There is some evidence that recombinational "hot spots" and homologous but unequal crossing over, leading to gene duplication and deletion, con- tribute to copy number polymorphism within the MHC (41). However, there are still a relatively small number of MHC genes compared with the number of potenUal antigens (c.f. Ig genes). A likely explanation for the low MHC gene copy number with extreme polymorphism probably lies in the function of the MHC molecules themselves and the processes by which T- cells are "educated" in the thymus to self MHC. The large number of MHC alleles means that a high proportion of individuals wiU be heterozygous at MHC loci; heterozygosity at MHC loci actually approaches 100% in outbred populations. Heterozygosity results in an overlapping set of MHC restriction elements that would increase the likelihood of recognition of any anUgen that could be encountered. A possible consequence of extensive MHC gene duplication is that a large number of polymorphic MHC products could lead to a preponderance of "blind spørs" in the antigen-specific T-cell repertoire (37).

For MHC genes, allele frequencies differ within and between racial groups and breeds of animals (41). What then influences the frequency of individual MHC alleles? Evidence that natural selecfion plays a major role in determining MHC allele frequencies can be found in studies of humans and animals (41). In humans, HLA gene frequencies were compared in the current Dutch population and a popula- tjon of Dutch that settled in Surinam in 1845 (86). The descendents of the Surinamese Dutch, who survived epidemic outbreaks of typhoid and yellow fever, had significant differences in the frequencies of HLA-A, HLA-B, and HLA- DR antigens from the present Dutch popula-

tjon. In chickens, the B21 haplotype, which contains specific resistance alleles for Marek's disease, is highly frequent in certain geographi- cal locations where the disease is endemic (66). Resistance to Marek's disease can be achieved simply by selecting for the B21 haplotype, without exposure to the Marek's disease virus (53).

Ånother line of evidence that natural selec- tjon plays an important role in determining MHC gene frequencies is the observation that certain MHC alleles are in linkage disequilibrium, i.e., alleles at different loci occur in combinafion more frequently than expected from their respective gene frequencies at the populafion level based on the Hardy- Weinberg equilibrium (10). In molecular terms, linkage disequilibrium of alleles at the class II loci can be explained by the apparent differences in pairing efficiency between a- and [~- chain aUomorphs (17). Selection would favør those allelic combinations that could fonn functional heterodimers.

Other hypotheses suggested to explain the maintenance of MHC polymorphism include "frequency dependent selection" and "defense against lateral transmission of pathogens" (6). These hypotheses assume that pathogens hare be.en selected for their ability to infect high frequency MHC types (antigen mimicry of the høst). Under these conditions, a large number of MHC alleles at low to moderate frequency would be favored. The latter hypothesis suggests that MHC polymorphism is a consequence of the ability of certain types of pathogens, e.g., retroviruses, to acquire høst antigens when they "bud" from infected cells. In order for the next horizontaUy infected høst to respond efficiently to such agents, an MHC difference would be required. This mechanism could only operate early in infection, because after infection the virus would acquire antigens of the new høst.

Artificial selection probably has a profound influence on MHC gene frequencies in dømes- tic animal species. The small number of found- ing sires and dams of a particular breed would limit the number and frequency of MHC alleles and haplotypes. Because of close linkage of the MHC loci and the relafively short history of cattle breeds, "linkage disequilibrium" in cattle could just as easily be explained by founder

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CLASS

REGION

LOCI

II II II I I I I I

DY DQ DR

I I I I I I I c 4 '1 I B A l l , , , , , , , , , , , , , , , , , , , , , ,

I I , i , , i , i , , i i i i . i i i i ,

Figure 1. Genetic map of the bovine major histocompatibility complex (BoLA). Abbreviations are BL complement factor B; C4, complement factor (24; 21-OH, steroid 21-hydroxylase (cytochrome P-450). The number of genes ha each region may vary in different haplotypes. The number, order, and relative map position of genes shown for the BoLA system are tentative and based on data from references (3, 4, 5, 7, 19, 70, 89); and Teulsch and Lewin, unpublislaed dala, for mapping of the BF lotus.

effect (gametic association) as by natural selection. Alternatively, selection (by humans) for a particular tralt (e.g., growth, milk production) that has major genes linked to the MHC would result in coselection of certain MHC haplotypes. The pøssible consequences of this are discussed.

PRESENT STATUS OF THE BOVlNE LYMPHOCYTE ANTIGEN SYSTEM

Current knowledge of the number and organization of bovine lymphocyte antigen (BoLA) genes and the structure and polymorphism of BoLA gene products has been obtained from serological, histogeneUc, biochemical, and molecular genetic studies. The BoLA classes I, II, and III antigens are all encoded on the short arm of bovine chromosome 23 (26) (Figure 1). Thus, as in other species, genes of the bovine MHC have been maintained as a conserved linkage group (41). The precise order of genes in the complex is not yet known, and the size of the BoLA region has yet to be determined. Data presented by Andersson et ai. (5) suggest that the BoLA complex may be relatively large since the genetic distance between BoLA-DQ and another class II locus, named -DY, was es- timated to be 17 centimorgans. Alternaåvely, a recombination hot spøt might exist between these loci, resulUng in distortion of the genetic map as has been found in the class II regions of the HLA and H-2 complexes (79).

Clase I Gønes and Gene Prod.cts

Using an HLA class I cDNA probe, at least 10 bovine class I genes were revealed by Southern blot analysis (85). Recently, two class I cDNA clones were sequenced from a bovine

B lymphoblastoid cell line (25). Sexluence variation in the 3' untranslated regions of these clones strongly suggests that the two clones represent products of different class I loci. Re- cent biochemical studies support that there are at least two class I molecules (12, 90), encoded by closely linked genes (90), that are expressed by lymphocytes. The organization, function, expression and polymorphism of the other BoLA class I genes detected by Southern blot analysis are not known.

Products of the BoLA-A (class I) locus have been extensively characterized using the serological approach (8, 19, 74). Alloantisera that distinguish BoLA-A allomorphs are readily obtained from primiparous or multiparous cows (the cow produces antibodies against the "foreign" paternal haplotype carried by the fetus), by skin grafting, or by deliberate immunization with lymphocytes. The most recent international workshop on the BoLA system, held in 1986, involved the exchange of 282 BoLA typing antisera between 20 laboratories in 13 countries, which were tested against a panel of 1298 cells from over 50 Bos taurus and Bos indicus breeds (19). This workshop produced agreement on 31 serologically defined specificities encoded by a single locus called BoLA-A. Included in the list of newly defined BoLA specificities were a number of new subtypes (also known as splits) of previously identified alloantigens. The frequencies of BoLA-A alleles varied greatly between breeds (19).

Until the most recent international BoLA workshop, there was scant evidence for a second polymorphic class I locus (77). Serological data suppørting the existence of a second polymorphic class I allelic series was presented in

Journal of Dairy Science VoL 72, Nø. 5, 1989


the workshop report (19). The new specificity, BoLA-w25, was associated with BoLA-w9 in some breeds and BoLA-w7 in others. Confir- mation of BoLA-w25 as a BoLA-B locus specificity awaits family studies.

A very sensitive technique recently devel- oped for HLA typing, called one-dimensional isoelectric focusing (1D-IEF), has demonstrated the existence of at least two polymorphic bovine dass I gene products on the lymphocyte cell surface (36, 90), which appear to be encoded by two tighfly linked loci (90). Interest- ingly, the putaUve BoLA-B locus specific bands were much less intense than the BoLA-A bands suggesting that BoLA-B may be expressed at a much lower level than BoLA-A (90). These results provide a possible explana- non of why it has been so difficult to detect BoLA-B products on lymphocytes using alloantisera. If true, the low expression of BoLA-B would be analogous to the lower expression of HLA-C relauve to the HLA-A and HLA-B gene products. Alternatively, some differences in band intensity observed in IEF experiments could be due to differences in affinity of the monoclonal antibodies for the different dass I gene products (36).

C l e u II G ø n u ond Gene Products

Histogenetic techniques were the first used in catfle to demonstrate the existence of antigens with the properties of dass II molecules, and the locus responsible for this reactivity, termed BoLA-D, was shown to be linked to BoLA-A (84). Monoclonal antibodies to phylo- geneticaUy conserved monomorphic epitopes on class II molecules were later used to directly reveal the presence of class II antigens on bovine B lymphocytes (23, 33, 47). Polymor- phism in BoLA class II gene products has prøven to be more difficult to define with serological reagents, but some attempts have been made (54, 62). Recently, the existence of at least two BoLA°A-linked polymorphic class I1 13-chains was directly demonstrated using 1D- IEF (89). Only one polymorphic class II 13- chain was expressed on activated T-cells, whereas two distinct polymorphic class II [3- chains (M~ 27,000 and M~ 28,000) were expressed on the surface of B-ceUs. Cattle aiso appear to have an unusual nonpolymorphic 13- chain expressed only in the cytoplasm.

Although the polymorphism of the BoLA class II molecules is only now being elucidated, the polymorphism of BoLA class II genes has been studied in detail using Southern blotting and restriction fragment length polymorphism (RFLP) analysis (3, 4, 5, 7, 70). The BoLA class II loci were named according to their human cøunterparts because human class II probes were used in the hybridization experiments. SegregaUon of RFLP patterns in paternal half-sib families has revealed the presence of three class II loci, termed BoLA-DR, BoLA- DQ, and BoLA-DY, each with at least two genes [ (3, 4, 5, 7); Figure 1]. Genes homologous to human DØl3 and DZct were also identified (5, 7). There is one BoLA-DR~ gene with five allelic patterns and at least three DR13 genes that have 25 associated RFLP types (3, 70). Molecular cloning and sequencing of a polymorphic DRI3-1ike pseudogene was recently reported by Muggli-Cockett and Stone (61). The number of BoLA-DQ genes varied between haplotypes; some haplotypes have one DQ~ and one DQ13 gene, but others carry a duplication of the entire DQ region (7). The DQa and DQ13 genes are highly polymorphic, as there are 20 allelic RFLP pattems correspond- ing to DQ~t and 17 RFLP pattems associated with DQ13 (4, 70). The two "novel" genes with more limited polymorphism, DY~ and DY13, were detected as cross-hybridizing fragments using human DQo~ and DR~ probes, respectively (5). Ahogether, a total of 30 DQ-DR haplotypes were detected in a sample of 197 breeding bulls and five sire half-sib families, comprising 50 offspring and 48 dams, of the Swedish Red and White breed.

The polymorphism of BoLA dass II genes is probably greater than what has been described. As more cattle breeds are studied, new allelic class II RFLP patterns will probably be found (61). As an example, we have found a DQot aUelic pattern in crossbred Simmental x (Angus x Hereford) cattle that has not previously been reported (Figure 2).

An obvious question is whether the gene polymorphism detected by RFLP analysis will be reflected at the protein level. Based on studies in humans, RFLP will be correlated with polymorphism at the structural level (87). How- ever, not all RFLP are expected to have a protein equivalent, because RFLP can result

Journal of Dairy Science Vol. 72, Nø. 5, 1989

1340 LEWlN

kb

9.0

5 .6

4 .8

2 .5

Figure 2. Hybridization of hovine genomic DNA with an HLA-DQOt probe. Genomic DNA from a Simmental x (Angus x Hereford) calf (BoLA-w6/w30) was digested with the restrictJon enzyme PvulI, electrophoresed in a .8% agarose g¢l, and transferred to a Bio-Rad Zeta Probe nylon membrane. The DNA was hybridized with a [azP]-labeled, 584 base pair Rsal/StuI fragment of a human DQ¢t cDNA sequence as described by Andcrsson et ai. (4). Hybridizing sequences were visualized by autøradiography after a 48-h expøsure. Size standards (lamMa ladder) are shown on the left. The allelic pattems shown have not previously bœn reported.


from sequence variation in pseudogenes, noncoding introns, flanking sequences, and neutral nucleic acid substitutions. Conversely, there will be structural polymorphism not detected by RFLP because the base change(s) may not produce a detectable restriction site.

Over the next few years, additional sequence information on BoLA genes will be forthcoming and homologous probes will become available. Indeed, we have recently isolated clones encoding bovine DQct and DQ~ from a cDNA library constructed from the BL-3 lymphoblas° toid cell line (Xu and Lewin, unpublished). Use of homologous probes wiU permit higher conditions of stringency for hybridization with genomic DNA, which will probably simplify the interpretation of RFLP patterns. For example, in cattle, the reduced conditions of slringency used with human DR~ probes results in cross° hybridization with DQI3 fragments (5, 7). The use of homologous, locus-specific and exon- specific probes wiU also facilitate the large- scale mapping of the BoLA region by deletion mapping (64) and pulsed field gel electropho- retic techniques (30).

Stratøgies for Detection of Immune Response and Dlsøasø Reslstance Gønøs In Cattlø

The genetic control of immune responses has not been extensively investigated in cattle because reagents for class II typing have not been readily available. Moreover, it is quite difficult to idenåfy families or populations neco essary for obtaining meaningful data. Population studies are useful for detecting MHC associations, but family studies can provide an understanding of dominance relationships (83).

The statisfical methods for detecting disease association in human populations are generally applicable to studies of cattle but are not ideal for the most convenient experimental designs because of the relatedness between individuals that occurs within and between herds. Cattle are not truly outbred, and within a herd, groups of patemal and matemal half-sibs usually exist. Thus, an observed association between a "BoLA-A allele" and a disease at the popula- tjon level could actually be due to a haplotype effect. More sophisticated staustical analyses, taking into account age, farnilial relationships,


etc., are clearly necessary for an unbiased as- certainment of disease association with the BoLA system at the population levd.

Rather than being a handicap, the existence of large paternal half-sib families prøduced by A1, and matemai half-sibs prøduced by embryo transfer, provide a unique opportunity to study disease association in cattle. In general, well- designed family studies will result in a much clearer picture of the role of the MHC in immune responsiveness and disease resistance, and appropfiate mødels exist for determining if the møde of inheritance is dominant or reces- sive (72, 83). The internationaily standardized and relatively inexpensive BoLA class I serol- ogy can effectively by used for "haplotype marking" in families, obviating the need to type for class II aUeles, assuming close linkage. A problem might arise in analyzing two families that share identical BoLA-A aileles if one is interested in what might be a class II-linked effect. The problem is whether the same class I ailele can be associated with different class II aileles. This is especiaUy important when analyzing data across breeds. At this time, the number of BoLA haplotypes and the extent of linkage disequilibrium between class I and class II loci are not known; however, RFLP or 1D-IEF anaiyses of sire class II haplotypes is now feasible and data on linkage disequilibrium across the entire BoLA complex should be forthcoming in the very near future. Recently, Andersson et ai. (5) hare shown strong linkage disequilibrium between DR and DQ aileles by RFLP studies.

The impact of new reprøductive biotechnologies on disease association studies is likely to be very great, if we can afford to prøduce animals in this way for research. The production of cloned "lines" of cattle using nuclear transfer would permit evaiuaUon of immune responsiveness and disease resistance much the way it has been done in inbred strains of mice. This would greatly simplify the lives of bovine immunogeneticists!

Another important aspect that must be considered in approaching disease association in any species is the complexity of the disease of interest. Diseases that have a simple etiology will most likely result in the most clear-cut answers concerning MHC involvement. As such, the most well-understood diseases, in

terms of MHC-associated immunogenetic mechanisms, are viral diseases. The study of diseases such as mastitis, which have multiple causative agents, is less likely to lead to an understanding of the immunologicai mechanisms underlying a possible BoLA association. Specific tests for the presence of a pathogenic agent(s) should be readily available, the role of the specific organism in the disease well understood, and subclinicai stages of the diseases should be easily identified. Classification of subclinicai stages of an infection will permit the identification of true "resistance aileles" becanse those infected animals that do not develop advanced subclinical infections tan be distinguished from those infected animais that do develop advanced stages of the disease. Because clinical disease is likely to be multi- factorial, with stress, age and physiologicai status playing important roles, the absence of clinical disease should not necessarily be interpreted as genefic resistance to disease. Alterna- tively, a strong associaåon with a clinicai disease, such as the example of I-ILA-B27 and ankylosing spondylitis (relative risk approaches 100), is evidence of direct MHC involvement in disease susceptibility.

Evldencø for Immune Response Genes In Cattle

In the Norwegian Red breed, Lie (51) found high antibody responsiveness to human serum albumin (HSA) in sire families of young bulls, and later, in the same individuals, an associa- tjon between BoLA-w16 and BoLA-w6 and high responder status (52). Bulls with BoLA- w2 were low responders to this antigen. Bulls with BoLA-w8 and BoLA-w20 were higher antibody responders to the branched synthetic peptide antigen (T, G)-A--L than animals with other BoLA-A specificities. Presumably, these associations are due to the effects of the closely linked class II genes. Interesungly, BoLA-A heterozygotes were significantly better responders than putative homozygotes. Among half sisters of these bulls, high responders to HSA (associated with w16) were susceptible to mastitis, whereas low responders (associated with w2) were relatively resistant to mastitis (73). Further evidence for an association between w16 and susceptibility to mastitis was suggested by earlier work of Larsen et ai. (42)


1342 LEWlN

in which an associafion between mastitis susceptibility and the M' allele of the M blood group was reported. The M' allele is highly correlated with the presence of BoLA-w16 and BoLA-A and M are either identical (32) or tighfly linked (44).

That bovine class I molecules act as restriction elements for CTL was demonstrated using alloreactive T cell clones (81). Class I restricted CTL clones specific for the intracellular protozoan parasites Theileria parva and T. annulata have also been produced (27, 67) and these cytotoxic cells (heterozygous for BoLA-A) show a strong preference for one allele of BoLA-A matched parasitized targets (60). These results might hare important implications for the future development of a T. parva (the agent of East Coast fever) vaccine (60).

As can be deduced from the cited studies, work on the genetic control of immune responsiveness in cattle is still in its infancy. The future widespread use of embryo transfer and embryo splitting and the newer technology of nuclear transfer for cloning cattle, wiU allow us to evaluate more effectively the importance of the BoLA system in immune responses to pathogens and resistance to disease.

Dlseasø Associatlons

Associations between BoLA class l antigens and tick infestation, worm infestation, ocular squamous cell carcinoma, and bovine leukemia virus infection were studied by Stear and his coworkers (75). In two independent experiments with 3/4 Brahman-1/4 Hereford cattle, some evidence for an association between BoLA-w16 (local specificity CA27) and reduced numbers of the tick Boophilus microplus was found (75). Susceptibility was associated with BoLA-w6 (CA2) in both experiments, but results were significant at 5% in only one experiment. In another study, evidence for an association between BoLA-w16 and lowered fe- cal worm egg counts (primarily Cooperia species and Haemonchus placei) was found among the offspring of a BoLA heterozygous sire (75). These results were not confirmed in Africander x Hereford cows; however, in this study BoLA-w9 and BoLA-CA45 were margin- ally significant for resistance and susceptibility, respectively (78). In a study of ocular squa-

mous cell carcinoma in Hereford cattle, a disease that has moderate heritability, the relative risk of cattle with the antigen BoLA-w6 for the disease was 8.5. However, when corrected for the number of comparisons made, the results were no longer significant at 5% (75). Some evidence was also presented for the involvement of BoLA-w6 and Eu28R in susceptibility to development of persistent lymphocytosis (PL) in IUawarra Shorthorn and a small number of Jersey and Holstein cattle infected with the bovine leukemia virus (BLV) [ (76) for a further discussion of BLV see next section]. The antigen BoLA-w8 was less frequent in the Shorthorn cattle with persistent lymphocytosis. The associations described for BoLA-w6 should be interpreted with caution, since these are now accepted as BoLA specificities with multiple subtypes (19).

Enzootic Bovine L.øuko$iø: A Model System

Enzootic Bovine Leukosis (EBL) is an eco- nomically important disease of cattle (20). In the United States, losses due to the terminal tumor stage of BLV-infection and restrictions on the export of BLV-infected animals and semen from BLV-infected bulls have been esti- mated at 44 million dollars per annum (82). Because the tumor phase of the disease devel- ops in only .5% to 3.0% of BLV-infected cattle (20), the aue cost of BLV infection, including all subclinical stages, might be much greater. Our recent work on the effect of subclinical BLV infection on milk production in Holstein cows demonstrated a dramatically lower fat percentage in milk of BLV-infected cows with PL as compared with uninfected cows (92). In light of these recent findings, and the high prevalence of BLV infection in US dairy herds, the full economic impact of BLV infection on the dairy industry needs to be reevaluated.

Enzootic Bovine Leukosis provides an ex- cellent model for genetic studies because BLV infection has several distinct subclinical stages (Figure 3). In our experiments (45, 46, 50), we asked whether BoLA was associated with the presence or absence of antibodies to the major BLV envelope glycoprotein (BLV-gp51), which is the most widely used diagnostic tool for BLV infection (20). In order to examine the possible role of BoLA in subclinical progres-

Journal of Dalry Science Vol. 72, Nø. 5, 1989


NON- INTEGRATION LYMPHOSARCOMA

EXPOSURE ) INTEGRATION- ) B-CELL ) INVERSION OF ( ) PL (provirus) PROLIFERATION T/B RATIO

ANTIBODIES - - +/- TO BLV-gp51

+ +

ANTIBODIES - - - + / - +

TO BLV-p24

EFFECT ? ?

OF BoLA + +

Figure 3. Model of the pathogenesis of bovine leukemia virus (BLV) infection. The mødel proposed is based oil experimental data from this and other labøratories. The frequency of exposed animals that become productively infected with BLV is unknown. The B cells are the primary target of BLV infection (20), but macrophages can also be infected (Lewin, unpublished results). Infected animals have BLV integrated into their DNA, which is termed BLV provirus (20). Usually within 1 yr, BLV-infected animals develop antibodies to the major viral envelope glycoprotein (BLV-gp51). The source of antigen (replicating virus) has not been determined but may be dividing B-cells or macrophages. Up to 70% of all BLV-infected animals have B-cell expansion that tan be detected by an increase in the raåo of B to T lymphocytes in peripberal blood (49). These B-cells are positive for surface membrane immunoglobulin M and class II antigens. Approximately half of the animals with increased B-cells (30% of BLV-infected animals) develop persistent lymphocytosis, (PL) which is defined by an increase in the absolute numbers of all lymphocytes in peripheral blood (55). The percentage of B lymphocytes in peripheral blood of PL cattle is usually in the range of 60 to 90%, whereas B-cell percentage in normal cattle ranges from 15 to around 50% (49). The PL stage of infection is correlated with the appearance of antibodies to the viral core protein p24. Tumors (lymphosarcoma) develop in less than 1% of infected cattle; however, this number may be affected by culling since animals diagnosed with lymphosarcoma have a mean age of 7 yr. Not all animals that develop tumors have had a history of PL, but inverted T:B ratios identify the total population at risk for developing lymphosarcoma (Lewin, unpublished). Steps in the pathogenesis where the BoLA system appears to have an effect on subclinical progression of BLV infection are shown.

sion of BLV infection, we used total lymphocyte count (55), percentage of B-cells, and absolute B-cell count in peripheral blood as parameters. Peripheral blood B-cell percentage and absolute B-cell concentration are useful parameters to monitor because B-cells are the primary target of BLV infection and increased B-cell percentage and absolute numbers were clearly shown to be correlated with subclinical progression of the disease (49). The very low incidence of tumors, relative to seroconversion and B-cell lymphocytosis, limits the study of genetic factors in tumor susceptibility. Howev- er, some preliminary data on the relationship between BoLA and lymphosarcoma have been reported (76, 80).

Results of two population studies, one in Illinois and one in California, demonstrated a significant negative association between BoLA- w8.1 [BoLA-wl4 according to new nomencla-

ture (19)] and the presence of antibodies to BLV-gp51 in Holstein cows (45, 50). In a recent prospective California study of Holstein cows (65), these findings were not confirmed; however, cows positive for BoLA-w8.1 sero- converted at a significantly older age than cows with other BoLA-A alleles. These observations point out the need to compare herd mean age when evaluating seroconversion data because seroconversion of BLV-gp51 is highly age-dependent (20, 49). Our present interpretation of these data is that cows with BoLA-w8.1 are not more resistant to BLV infection per se. The relative age-related resistance of w8.1-positive cows to seroconversion and B-cell expansion suggests that a closely linked gene(s) might act to slow the spread of BLV early in infection, or simply that animals with w8.1 are low responders to BLV-gp51. As with most retroviruses, BLV infection persists even in the presence of

Journal of Dalry Science Vol. 72, Nø. 5, 1989

1344 LEWIN

high fiters of neutralizing antibodies. Hence, the biological significance of this association, if any, has yet to be determined.

In the two Holstein herds that we have studied, BoLA-wl2.1 [BoLA-wl2 according to new nomenclature (19)] was associated with an increased frequency of lymphocytosis and increased percentages and absolute numbers of B- cells among BLV-infected cows (45, 50). In both herds, BLV-infected cows that were w8.1 ÷ tended to have a lower frequency of lymphocytosis and lower numbers of B-cells in peripheral blood, The significant increase in B- cells and increased frequency of lymphocytosis among BoLA-w12.1 ÷ cows in independent herds demonstrates the existence of BoLA- linked genes that influence subclinical progression of BLV infection. In addition, our study of BLV infection in Shorthom cattle (46), and studies of others (65, 76), also support a role for the BoLA system in influencing the outcome of BLV infection, although different class I alleles had significant effects in the different breeds.

We have proposed that class II genes are responsible for the observed association between BoLA-wl2.1 and the susceptibility of BLV-infected cows to polyclonal expansion of B cells (48, 50). Our hypothesis is based on the following observations: 1) the association between w12.1 and B cell lymphocytosis is relatively weak at the population level (combined estimate of relative risk = 2.4), 2) different class I alleles had significant effects in the different breeds, 3) BLV is not expressed by BLV-infected cells in vivo (40), so that class I restriction elements cannot be involved in recognition of viral determinants by T lymphocytes, and 4) we have noted an unusual class II phenotype, the loss of BoLA-DR expression at the B cell surface, which is highly associated with PL and lymphosarcoma (48). One possible mechanism for a class II-linked effect is that the loss of BoLA-DR results in a failure of antigen presentation, which in this case might be a described tumor-associated transplantation antigen (1) or an immunoglobulin idiotype on expanding clones of BLV-infected B-cells. It will certainly be important to determine what effects provirus insertion and expression have on the transcription of BoLA genes.

A final interesting aspect of the relationship

Joulllal of Dairy Science Vol. 72, Nø. 5, 1989

between the BoLA system and BLV infection is the apparent longer herd life of the resistant cows and shorter herd lire of susceptible cows (46, 65). These differences were BoLA-associated and suggest that in herds with a high prevalence of BLV infecfion, BoLA-mediated resistance to BLV infection is an important component of longevity. Our recent study of the relationship of breeding values to susceptibility to BLV infecfion and the effects of BLV infection on milk production (92) supports the hypothesis that genetic resistance to subelinical progression of BLV infection has an important economic value. Long-term prospective studies by this laboratory and others are presently underway to define more precisely the role of the BoLA system in BLV infection and disease progression and their relationship to production traits and herd life.

IMPLICATIONS OF SELECTIVE BREEDING FOR ALLELES, MAJOR HISTOCOMPATIBILITY

COMPLEX BREEDING, AND FUTURE APPLICATIONS

The possibility of using BoLA as a selection tool to increase resistance to a specific disease needs careful considerafion. Would selection for resistance to one disease make cattle more suscepUble to other infecfious agents? Work by Biozzi et ai. (13) with mice suggests that this might occur. In the cheetah, limited MHC polymorphism, possibly the result of a population "bottleneck", has been suggested as the reason for the susceptibility of the remaining population to feline infectious peritonitis (63). How- ever, chickens selected for resistance to Ma- rek's disease are, to a large extent, homozygous for the B21 haplotype (66) and do not seem to have a higher incidence of other diseases. As discussed, natural selection has favored extensive MHC polymorphism in most species, and extreme changes in MHC gene frequencies could have drastic consequences. Selection against susceptibility by "haplocide", if the haplotype is present at a low frequency, might be warranted under certain conditions (e.g., lack of an effective vaccine, no desirable traits associated with that haplotype). Alterna- tively, BoLA-dependent selection for general fitness, as measured by longevity in a given environment, could also be a successful and safe strategy. The essential point in any selec-


tjon program utilizing properties of the MHC is to maintain heterozygosity. It certainly wiU be most interesUng to determine the effect that selective pressure for milk producUon has bad on MHC polymorphism in dairy breeds that have relatively higher incidence of reproductive diseases and mastitis.

What can be gained from knowledge of BoLA-l inked immune responsiveness and resistance or susceptibility to infectious diseases? First, some diseases of cattle, such as bovine leukosis, are exceUent models for studying mechanisms of human diseases. Recent atten- tion has be.en focused on BLV because of its close relafionship to a cancer-causing retrovirus in humans (21). A decided advantage of any animal model is that challenge with pathogenic organisms, isolation, repeated sampling, and selective breeding can be done. It is relatively certain that touch will be learned about the immunological mechanisms of model diseases and economical ly important diseases from the study of the bovine MHC. Such knowledge might be used for therapeutic intervention when specific mechanisms of the disease process are known.

Another interesting application is the production of Iransgenic animals with "resistance alleles" to specific diseases (88). For infectious diseases for which there are nø effecuve vaccines, such as BLV and mastitis, identification, doning , and transfer of MHC allomorphs that confer relative resistance to infection or disease would be highly desirable. It is appropriate to note that tissue specific expression of class II genes in transgenic mice has been achieved (43).

Perhaps the most exciUng and challenging possibi l i ty will be to use knowledge of MHC polymorphism to produce more effecUve vaccines. This will require large-scale purification and extensive knowledge of the sequences of bovine class II alleles so that the relative binding efficiencies and antigen presenting ability of the different BoLA allomorphs can be determined. Information on immunodominant struc- tures and neutralizing sites on viruses, bacteria, parasites, and the chemical mediators that they produce will also be needed. Efforts to produce "designer pepUde" vaccines are now underway in humans. It is only a matter of time before this type of research will be initiated in cat- tie.

ACKNOWLEDGMENTS

The technical and scientific work of W. C. Wu, J. A. Stewart, T. J. Nolan, and J. E. Beever on the BLV studies is gratefully acknowledged. The author would also like to thank K. W. Kelley and L. B. Schook for their helpful com- ments on this manuscript.

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Disease Resistance and Immune Response Genes in Cattle: Strategies for Their Detection and Evidence of Their Existence